Recent decades have seen a progressive miniaturization of high-performance liquid chromatography (HPLC) systems, in particular with regard to the mobile phase flows utilized. This is because flow reduction particularly enables the consumption of costly toxic solvents to be limited; it also results in a reduction in injection volumes, and hence enables very small sample quantities to be processed. In addition, if concentration-sensitive sensors are used, a sensitivity increase results because of the smaller dilution of solvent in the column and a better signal-noise ratio.
Finally, the new liquid chromatography-mass spectrometry (LC-MS) coupling techniques enable working at flows less than 1 μL/min, to obtain higher sensitivity and limit possible contamination risks.
However one of the problems encountered when working at very low flows consists of the lack of instrumentation able to generate reliable mobile phase gradients, with only a small delay and homogeneous mixing of solvents. The generation of a gradient at flows less than 1 μL/min certainly requires an adequate pumping system, able not only to carefully and homogeneously mix the mobile phases, but also to transfer them into the column in a reproducible manner and with minimum delay. However, under these conditions the commercially available instrumentation is compelled to operate at its limit, and hence the performance reliability is often questionable. Moreover the effective gradient profile presents an unsatisfactory pattern compared with that set.
One of the most common ways of generating micro- and nanoflow mobile phase gradients is the split-flow technique using a splitter inserted between the HPLC pumps and the injector. This solution is limited by poor control of the gradient actually transferred into the column, and consequently of poor reproducibility. One of the initial attempts to achieve nano-scale gradients was to store a specific gradient in a capillary and to then transfer it into the column with a syringe pump. Drawbacks of this system are poor practicality, a limited choice of gradients to be formed, and poor control of the shape of the gradient.
Another known system, known as the exponential dilution method, consists of rapidly passing the mobile phase flow from the weak eluent, contained in a mixing chamber, to the strong eluent, with generation of a gradient of concave exponential profile.
The drawback of this system is the limited number of gradient types which can be generated.
To overcome this drawback, it has already been proposed to fill the mixing chamber with the weak eluent and to cause the strong eluent to flow in a programmed manner into a dynamic mixer, in order to be able to control the gradient variation with time.
The drawback of this system is a poor versatility of achievable gradient profiles.
Another known system consists of generating an exponential nanoflow gradient by introducing into two successive mixing chambers firstly the weak eluent and then the strong eluent, and then transporting them into the column using one pump at a time.
The drawback of this known solution consists of a limited gradient and profile selection, in addition to a consistent gradient delay.
U.S. Pat. No. 7,135,111 describes a nanoflow gradient elution device. It comprises a first pump for mixing different solvents and transporting them, a second pump for transporting a transfer solution, an injector, a column and a detection system. Two loops enable different solvent mixtures to be temporarily stored, these being transferred by the first microflow pump and then transported into the nanoflow column by the second pump, by a modification in the internal system connections and hence in the flow direction.
The difficulty of this known solution is the difficulty of delivering the mobile phase at constant flow when working with nanolitres/min.
U.S. Pat. No. 7,141,161 describes a gradient pump arrangement able to transfer eluents to a chromatograph continuously, at specific time intervals and at a constant flow of the order of nanolitres/minute while the composition of two or more eluents is modified. The gradient pump arrangement includes a ten-port multi-position valve, a first loop connected to an isocratic pump and a second loop, through which the eluents are transferred.
The drawback of this known solution consists of a considerable complexity of the pump and valve system, making it particularly difficult to automate the instrumentation.
From Cappiello A. et Al “Variable-Gradient Generators for Micro and NanoHPLC”, Anal. Chem. 2003, 75, 1173-1179 a device is known for generating micro- and nanoflow mobile phase gradients for liquid chromatography.